Protein aggregation and misfolding play major roles in protein production in the biotechnology industry, in limiting the biochemical study of proteins, and in the onset of pathogenesis in human disease. The native, correctly folded state is necessary for a protein's biological function and recognition by other molecules; misfolding and misassembly lead to significant loss of biological activity. Because the mechanism that drives aggregation is poorly understood it represents a challenge to industrial, academic, and medical research scientists.
Aggregation can occur by many distinct mechanisms (De Bernardez Clark, 1998). One such mechanism is formation of intermolecular disulfide bonds (e.g., Stoyan et al., 1993). Currently, refolding of proteins that have aggregated by incorrect disulfide bonding has been achieved by addition of oxidants or redox buffers (Builder et al., 1997; De Bernardez-Clark and Georgiou, 1991; Rudolph and Lilie, 1996). However, in many cases, other mechanisms predominate. For example, the aggregation events that result in Alzheimer s disease and prion diseases such as Creutzfeldt—Jacob's disease and bovine spongiform encephalopathy are believed to occur by noncovalent association of β-strands (Bychkova and Ptitsyn, 1995; Jarrett et al., 1993; Thomas 1992, 1995). Aggregation of tailspike protein is also believed to result from specific association of partially folded chains, possibly by misalignment of the β-strands (Speed et al., 1996).
Current methods for refolding proteins from non-covalent aggregates and inclusion bodies typically require that the proteins first be solubilized and nearly completely unfolded, typically through the use of strong chaotropic agents, such as guanidine chloride or urea, and reducing agents (Cleland, 1993). The proteins are then refolded by removal of the denaturant.
Aggregation is favored at higher protein concentrations. Consequently, removal of the denaturant frequently requires large dilution and therefore large working volumes. Low refolding yields are therefore common, due to the loss of protein during refolding and subsequent concentration (De Bernardez-Clark and Georgiou, 1991). Yield of protein in its native conformation upon renaturation is often low, regardless of refolding conditions.
To help prevent aggregation, refolding is often performed stepwise, using a series of gradual buffer changes, which decrease the concentration of a chaotropic denaturing agent. A second existing method to inhibit aggregation during refolding is to elevate, then slowly decrease temperature. Temperature control is also used to disfavor aggregation. Both of these processes are slow, labor intensive, costly and inefficient.